The immunoinhibitory receptors PD-1 and TIGIT play critical roles in regulating tolerance, and are key mediators of T cell dysfunction in cancer and chronic infections. Anti-PD-1 antibody is now an FDA approved therapy for cancer, while anti-TIGIT is a highly promising target for enhancing anti-tumor immunity. While PD-1 blockade is having impressive therapeutic effects in some cancers, many patients do not respond to single agent immunotherapy. With recent studies showing striking synergies between the PD-1 and TIGIT pathways in regulating T cell dysfunction in vivo, combination therapies are under consideration. Our lab along with our collaborators in this PPG have shown that PD-1:PD-L1 interactions regulate both the induction and maintenance of peripheral T cell tolerance, and that TIGIT has T cell intrinsic inhibitory effects that regulate tolerance. However, little is known about the mechanisms by which these inhibitory pathways work individually, and particularly in combination, to inhibit T cell activation. The goal of Project 3 is to determine the interactions between TIGIT and PD-1 in controlling CD4+ regulatory T cells (Treg) and CD4+ FoxP3? T cells, and define the genetic circuits underlying these interactions. In the first aim, we will test the hypothesis that interactions between TIGIT and PD-1 control Treg function and/or stability, and synergize to have a dominant effect on CD4+ FoxP3- T cells, thereby controlling T cell tolerance. We will examine how combinations of agonists or antagonists to PD-1 and TIGIT alter the function of Treg and self reactive CD4+ FoxP3? T cells in vitro, as well as the pathogenicity of myelin-reactive CD4+ T cells in vivo in the EAE model. We will use TIGIT/PD-1 double conditional knockout mice (cKO) to test synergy within specific cell types. In the second aim, we will infer the transcriptional networks mediating the actions of PD-1 and TIGIT in Tregs and CD4+ FoxP3? T cells, based on the dynamic profiles of RNA expression in activated CD4+ FoxP3? T cells and Treg, and measurements of open chromatin by ATAC-seq. Guided by this inferred transcriptional map, we will systematically perturb and test the functions of candidate transcription factors in vitro during T cell activation or in vivo upon transfer of MOG-specific T cells to induce EAE. The results of this proposal should provide critical insights into how TIGIT and PD-1 interact to modulate T cell activation and tolerance. Furthermore, our results will contribute directly to Projects 1 and 2 by providing explanations for the changes observed in T cell functions in the absence of TIGIT (Project 1) and functional differences in T cells between MS patients vs. malignant gliomas based on PD-1 and TIGIT expression and activity (Project 2). Improvements in our functional and mechanistic understanding of costimulator and coinhibitor interactions should lead to new therapeutic targets and strategies for reducing autoimmunity while increasing anti-tumor immunity.